Zinc fingers belonging to the Cys2-His2 family constitute one of the most common DNA-binding motifs found in eukaryotes, and these zinc fingers have provided a very attractive framework for the design and selection of DNA-binding proteins with novel sequence specificities. Numerous studies have used phage display methods or design ideas to explore and systematically alter the specificity of zinc finger DNA interactions (Desjarlais & Berg, Proteins Struct. Funct. Genet. 12:101-104 (1992); Desjarlais & Berg, Proc Natl. Acad. Sci. USA 90:2256-2260 (1993); Rebar & Pabo, Science 263:671-673 (1994); Jamieson et al., Biochemistry 33:5689-5695 (1994); Choo & Klug, Proc. Natl. Acad. Sci. USA 91:11163-11167 (1994); Wu et al., Proc. Natl. Acad. Sci. USA 92:344-348 (1995); and Gresiman & Pabo, Science 275:657-661 (1997)).
Structure based computer design has been used to link Cys2-His2 zinc fingers with other DNA-binding domains, including other zinc finger proteins, to generate hybrid proteins that recognize extended sites (Pomerantz et al., Science 267:93-96 (1995); Kim et al., Proc. Natl. Acad. Sci. USA 94:3616-3620 (1997)). For example, zinc finger proteins have been linked to a GAL4 dimerization domain to develop novel homo- and hetero-dimers (Pomerantz et al., Biochemistry 4:965-970 (1997)), and to a nuclease domain to generate novel restriction enzymes (Kim et al., Proc. Natl. Acad. Sci. USA 93:1156-1160 (1996)). Zinc finger/homeodomain fusion is being tested for potential applications in gene therapy (Rivera et al., Nature Med. 2:1028-1032 (1996)).
There also have been several attempts to increase affinity and specificity of zinc finger proteins by adding additional fingers to a three-finger protein (Rebar, (Ph.D. Thesis), Selection Studies of Zinc Finger-NA Recognition, Massachusetts Institute of Technology (1997); Shi, Y. (Ph.D. Thesis) Molecular Mechanisms of Zinc Finger Protein-Nucleic Acid Interactions, Johns Hopkins University (1995)) or by tandemly linking two three-finger proteins (Liu et al., Proc. Natl. Acad Sci. USA 94:5525-5530 (1997)). However, these previous design strategies for poly-finger proteins, which all used canonical “TGEKP” linkers (linkers having the amino acid sequence threonine-glycine-glutamate-lysine-proline) to connect the additional fingers, resulted in relatively modest increases in affinity. There is thus a need to develop linkers that provide enhanced affinity and specificity to chimeric zinc finger proteins.